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Method of Inhibiting Secretase Activity

Inactive Publication Date: 2007-11-29
LOCOMOGENE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] It is an object of the present invention to provide a therapeutic agent usefu

Problems solved by technology

However, although several candidate substances have been obtained, no secretase inhibitor has yet been successfully developed.

Method used

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  • Method of Inhibiting Secretase Activity

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0093] This example is a working example of Herp screening using the Yeast Two Hybrid system.

[0094] A MATCHMAKER system (CLONTECH) was used for the Yeast Two Hybrid system, which employed the yeast transformation method (Pro. Natl. Aca. Sci. USA, 88: 9578-9582, 1991).

[0095] The end of synoviolin cDNA from 706 bp (amino acid #236) to 1851 bp (amino acid #617) or from 805 bp (amino acid #269) to 1260 bp (amino acid #420) was inserted into the Eco R I / Xho I site of a pGBT9 vector as EcoR I / Xho I (from 706 bp (amino acid #236) to 1851 bp (amino acid #617) of synoviolin is hereinafter called Syno dTM, while from 805 bp (amino acid #269) to 1260 bp (amino acid #420) is called Syno Ring: see FIG. 1). In FIG. 1, “bp” indicates a position in the nucleotide sequence.

[0096] The library used for Herp screening consisted of human cartilage-derived cDNA inserted into a pACT2 vector (pACT2-Y). Following 15 minutes of heat shock at 42° C., pGBT9-Syno dTM or pGBT9-Syno Ring (2.0 μg) were inserted...

example 2

[0100] In this example, synoviolin was bound to Herp in vitro.

[0101] pcDNA3-Flag-Syno dTM was prepared with synoviolin from 706 bp to 1854 bp and a TNT-coupled Translation System (Promega) inserted, and subjected to in vitro translation to prepare (i) a Flag-Syno dTM fusion protein, (ii) a Herp GST fusion protein, and (iii) a GST fusion protein with fragmented Herp (FIG. 2). In FIG. 2, “a.a.” shows the position in the amino acid sequence, while UBQ indicates a ubiquitin domain.

[0102] These proteins and (iv) GST protein as a control were added to a binding buffer comprising 20 mM Hepes (pH 7.9), 100 mM NaCl, 1 mM EDTA, 0.05% Tween, 5% Glycerol, 1 mM DTT, 0.2 mM NaVO4, 5 mM NaF and 1 mM PMSF, and reacted for 16 hours at 4° C. The reaction product was subjected to SDS-PAGE, and radioactivity was detected with an image analyzer (BAS2000, Fujix).

[0103] As a result, binding was observed between the Herp-M and Herp-C GST fusion proteins and the [35S]pcDNA3-FlagSyno dTM.

[0104] No bindin...

example 3

[0105] In this example, synoviolin and Herp were bound in vivo.

[0106] 2×106 HEK293 cells were inoculated on a 10 cm dish and cultured for 24 hours, and 3 μg each of pcDNA3-HA / Syno with synoviolin from 1 bp to 1854 bp inserted and pcDNA3-Flag / Herp with Herp from 1 bp to 1176 bp inserted were introduced into the cells. 24 hours after gene introduction the cells were collected and lysed with lysis buffer, and immunoprecipitation was performed overnight at 4° C. with anti-HA and anti-Flag antibodies. The bound proteins and free proteins were separated by centrifugation, and detected by Western blotting. Anti-HA and anti-Flag antibodies were used for detection.

[0107] As a result, a Flag-Herp band was confirmed by detection with anti-Flag antibodies following immunoprecipitation with anti-HA antibodies in the cells co-expressing synoviolin and Herp. Moreover, an HA-synoviolin band was confirmed by detection with anti-HA antibodies following immunoprecipitation using anti-Flag antibodies...

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Abstract

The present invention provides a method for inhibiting secretase activity. A method for promoting the sensitivity of a secretase inhibitor, a method for binding synoviolin to Herp (homocysteine-inducible endoplasmic reticulum stress-inducible ubiquitin-like domain member 1), or the like is employed to inhibit secretase activity.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for inhibiting secretase activity and a pharmaceutical composition thereof. Specifically, it relates to a method for inhibiting secretase activity by inhibiting synoviolin expression, thereby promoting the sensitivity of a secretase inhibitor, to a method for inhibiting secretase activity by binding synoviolin to Herp, and to a pharmaceutical composition comprising a substance that inhibits secretase activity. BACKGROUND ART [0002] Synoviolin is a novel protein that was discovered as a membrane protein in synovial cells derived from rheumatoid arthritis patients (WO 02 / 05207). Since studies using genetically modified animals have shown that this factor is directly involved in bone and joint development and in the development of joint disease, synoviolin is thought to be a protein whose activity contributes to normal bone formation or limb development. [0003] Because synoviolin is expressed ubiquitously not only in the ...

Claims

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Application Information

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IPC IPC(8): A61K38/55A61P25/28A61K31/7088A61K45/06C12N15/113
CPCA61K31/7088A61K45/06C12N15/1137C12N2310/14A61K2300/00A61P25/00A61P25/28A61P43/00
Inventor NAKAJIMA, TOSHIHIROAMANO, TETSUYAZHANG, LEIIKEDA, RIEYAMASAKI, SATOSHIYAGISHITA, NAOKO
Owner LOCOMOGENE INC
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